تسجيل الدخول

تسجيل

Interpretation of spectra and qualitative analysis

المؤلف:  Sue Jickells , Adam Negrusz (Editors)

المصدر:  Clarkes Analytical Forensic Toxicology

الجزء والصفحة:  p417-418

2026-11-11

72

Interpretation of spectra and qualitative analysis

For compounds with appropriate fluorescence properties, spectrofluorimetry gives high sensitivity and high specificity. High sensitivity results from the difference in wavelengths between the exciting and fluorescence radiation. This results in a signal contrasted with an essentially zero background; it is always easier to measure a small signal directly than a small difference between two large signals, as is done in absorption spectrophotometry. High specificity results from dependence on two spectra, the excitation and emission spectra, and the possibility of measuring the lifetimes of the fluorescent state. Although in biological samples the fluorescence intensity of interfering substances may be relatively high, the sensitivity and selectivity of the method are generally such that fluorescent drugs and their metabolites can be analysed more readily than by conventional spectrophotometry. Two compounds that are excited at the same wavelength but emit at different wavelengths are readily differentiated without the use of chemical separation techniques. Likewise, two compounds may fluoresce at the same wavelength but require different excitation wavelengths, thus enabling differentiation. Also, a fluorescent compound in the presence of one or more nonfluorescent compounds is readily analysed fluorimetrically, even when the compounds have overlapping absorption spectra. Nonfluorescent or weakly fluorescent compounds can often be derivatised with strong fluorophores, which enables them to be determined quantitatively. Many fluorescent species contain ionisable groups with fluorescent properties sensitive to pH. In somecases only one of the ionised species may be fluorescent. Examples are the barbiturates, which only fluoresce at an elevated pH in the di-anionic form. Phenol fluoresces at pH 7, but at pH 12, when it is converted into its anion, there is no fluorescence. Therefore, the relationship of fluorescence intensity with pH should always be examined as part of the method development.

Fluorescence intensity and wavelength often vary with the solvent. In most molecules, fluorescence decreases with a decrease in solvent viscosity as the probability of intermolecular energy transfer tends to be enhanced. The same effect occurs with an increase in temperature.